Oligotex Direct mRNA Kits enable purification of mRNA from cells and tissues. Each Oligotex Kit includes Oligotex resin for binding poly A+ mRNA, and spin columns for washing and eluting bound mRNA. For purification of mRNA from total RNA and cleanup of poly A+ in vitro transcripts, Oligotex mRNA Kits are available.

The Oligotex Direct mRNA Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Performance

Oligotex Direct mRNA kits are available for isolation of mRNA from as few as 100 cells to as many as 1 x 108 cells (see Table 1), or from less than 10 mg of tissue up to 1000 mg of tissue (see Table 2).

Poly A+ mRNA purified from tissues or cells is suitable for use in downstream applications such as RT-PCR (see figure "RT-PCR of beta-actin mRNA").

Table 1. Specifications of Oligotex Direct mRNA for purification of mRNA from cells

Table 2. Specifications of Oligotex Direct mRNA for purification of mRNA from tissues

Amount of starting material (tissue) per prep

Maximum number of preps per Oligotex Direct mRNA Mini Kit

Maximum number of preps per Oligotex Direct mRNA Midi/Maxi Kit

≤10 mg

12 (14)*

6 (14)*

10–25 mg

12 (14)*

6 (14)*

25–50 mg

6

6 (14)*

50–100 mg

3

6 (9)*

100–150 mg

–

6

150–250 mg

–

6

250–500 mg

–

4

500–750 mg

–

2

750–1000 mg

–

2

* Maximum number of preps per kit with purchase of additional spin columns. Additional 1.5 ml microcentrifuge tubes may be required.

Principle

Oligotex Direct mRNA Kits allow isolation of pure poly A+ mRNA directly from cells or tissues in as little as 1 hour (30 minutes with optional shortened protocols). The Oligotex Direct mRNA purification procedure utilizes rigorous denaturing lysis conditions to generate an immediate RNase-free environment for the isolation of intact mRNA. Oligotex Direct mRNA Kits contain a guanidine-thiocyanate–based buffer for efficient cell and tissue lysis, protein denaturation, and RNase inactivation, ensuring isolation of intact mRNA. The optimal design of Oligotex particles, in combination with RNA-protecting lysis and hybridization conditions, results in efficient purification of poly A+ mRNA in a short time with greater than 90% recovery. Poly A+ mRNA purified with Oligotex technology does not require further purification and is immediately ready for any standard application.

Oligotex resin consists of polystyrene–latex particles of uniform size (1.1 µm diameter) and a perfect spherical shape (see figure "Oligotex particles and oligo-dT cellulose") with dC10T30 oligonucleotides covalently linked to the surface. The size, composition, and surface structure of Oligotex particles have been optimized for uniform dispersion and minimal centrifugation time. The particles form a stable suspension that provides a large surface area for rapid and efficient binding of polyadenylic acids. The high capacity and accuracy of hybridization provides superior purification of poly A+ mRNA through fast and efficient hybridization.The Oligotex purification procedure minimizes loss of poly A+RNA, eliminates the risk of degradation by RNases, and requires minimal hands-on time. This makes it ideal for simultaneous handling of multiple samples.

Procedure

Optimized Oligotex purification procedures are convenient and straightforward (see flowchart "Oligotex mRNA procedure"). Homogenized cell or tissue lysates are incubated with Oligotex resin, and Oligotex:mRNA complexes are collected by a brief centrifugation step. Spin columns, supplied in the Oligotex Kits, provide the most convenient handling, and optional batch protocols are also provided for certain applications. After washing, the mRNA is eluted in a small volume of Tris buffer or water. Alcohol precipitation is not required.

Applications

Poly A+ mRNA purified with Oligotex resin is ready for use in any downstream application, including:

The procedure has been successfully used for isolation of mRNA from adipose tissue by customers. Because of the nature of adipose tissue it cannot be used immediately in Oligotex Direct mRNA Protocols; simple centrifugation, however, eliminates the problem, and the procedure works fine.